Production of Sialylated Oligosaccharides in Bacillus Cells

Abstract

Disclosed are non-sporulating Bacillus cells for the production of a sialylated oligosaccharide wherein said Bacillus cell has been genetically engineered to possess a lactose permease, a CMP-NeuNAc biosynthesis pathway and a sialyltransferase, as well as a method for producing a sialylated oligosaccharide using said Bacillus cell.

1 . A non-sporulating  Bacillus  cell for the production of a sialylated oligosaccharide, wherein said  Bacillus  cell has been genetically engineered to possess a lactose permease, a CMP-NeuNAc biosynthesis pathway and a sialyltransferase. 
     
     
         2 . The  Bacillus  cell according to  claim 1 , wherein the sporulation capability of the  Bacillus  cell has been impaired by deletion or functional inactivation of one or more of the genes encoding Spo0A, sigma E and sigma F. 
     
     
         3 . The  Bacillus  cell according to  claim 1  or  2 , wherein the lactose permease is  E. coli  LacY or a functional variant thereof. 
     
     
         4 . The  Bacillus  cell according to any one of  claims 1  to  3 , wherein the CMP-NeuNAc biosynthesis pathway uses GlcN-1P or GlcNAc-6P as intermediate. 
     
     
         5 . The  Bacillus  cell according to any one of  claims 1  to  4 , wherein the CMP-NeuNAc biosynthesis pathway uses a sialic acid salvage pathway. 
     
     
         6 . The  Bacillus  cell according to any one of  claims 1  to  5 , wherein the sialyltransferase is a lactose-accepting sialyltransferase, preferably a sialyltransferase selected from the group consisting of α-2,3-sialyltransferases, α-2,6-sialyltransferases and α-2,8-sialyltransferases. 
     
     
         7 . The  Bacillus  cell according to any one of  claims 1  to  6 , wherein said  Bacillus  cell lacks any β-galactosidase activity or possesses reduced β-galactosidase activity as compared to a wild-type progenitor  Bacillus  cell of the same species. 
     
     
         8 . The  Bacillus  cell according to  claim 7 , wherein the  Bacillus  cell has been genetically engineered by deletion or functional inactivation of at least one of the genes selected from the group consisting of yesZ and ganA. 
     
     
         9 . The  Bacillus  cell according to any one of  claims 1  to  8 , wherein said  Bacillus  cell is a  Bacillus subtilis  cell. 
     
     
         10 . The  Bacillus  cell according to any one of  claims 1  to  9 , for use in the production of 3′-sialyllactose, wherein the CMP-NeuNAc biosynthesis pathway uses GlcN-1P as intermediate, and wherein the sialyltransferase is a α-2,3-sialyltransferase. 
     
     
         11 . The  Bacillus  cell according to any one of  claims 1  to  9 , for use in the production of 6′-sialyllactose, wherein the CMP-NeuNAc biosynthesis pathway uses GlcN-1P as intermediate, and wherein the sialyltransferase is a α-2,6-sialyltransferase. 
     
     
         12 . Use of a  Bacillus  cell according to any one of  claims 1  to  11  for the production of a sialylated oligosaccharide. 
     
     
         13 . A method for the production of a sialylated oligosaccharide, the method comprising
 providing a non-sporulating  Bacillus  cell as defined in any one of  claims 1  to  11 ;   cultivating said  Bacillus  cell in a fermentation broth and under conditions that are permissive for the production of a sialylated oligosaccharide; and optionally   retrieving the sialylated oligosaccharide from the medium and/or the  Bacillus  cell.   
     
     
         14 . The method according to  claim 13 , wherein the fermentation broth contains lactose. 
     
     
         15 . A sialylated oligosaccharide produced by a method according to  claim 13  or  14 . 
     
     
         16 . Use of a sialylated oligosaccharide according to  claim 15  for the manufacture of a nutritional composition, preferably of an infant formula. 
     
     
         17 . A nutritional composition containing a sialylated oligosaccharide according to  claim 15 .